Detecting and Modifying Chromatin Organization in the Budding Yeast Genome with Synthetic Proteins

DNA in the nucleus of eukaryotic cells is condensed into chromatin by forming specialized structures called nucleosomes. DNA is wrapped around a set of histone proteins to from nucleosomes, which are eventually positioned by robust, reproducible mechanisms. Mutations in chromatin organizing proteins are common in human diseases, indicating that studying the systems in place to control chromatin organization could be insightful for developing novel therapeutics. S. cerevisiae is an ideal model organism for this work, as it is tolerant to dysregulated nucleosome positioning, genetically tractable, and contains homologs of many human proteins. Here, I describe several studies that have furthered our understanding of chromatin organization in the context of nucleosome positioning. First, I detail a set of synthetic proteins that enable nucleosomes to be repositioned at characteristic sites in the yeast genome. Following this is a detailed account of our investigation into the biochemical mechanisms underlying targeted chromatin remodeling at a subset of genome loci by the chromatin remodeler Isw2. Next, a protocol for efficiently detecting nucleosome positions is described, including its application to yeast deficient in chromatin remodeling activity. In chapter five, I present a system for detecting protein-DNA interactions and nucleosome positions using targeted nuclease digestion with endogenously expressed proteins. I also outline some of the potential applications of this technique, including use with chromatin remodeling proteins, which often prove difficult to map with standard methods.This dissertation contains previously published co-authored material.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Department of Chemistry and Biochemistry

Grantor dc:publisher

University of Oregon

Year dc:date.issued

2023

Author dc:creator

• Banks, Orion

Advisor dc:contributor.advisor

• Harms, Michael

• Chromatin
• DNA binding proteins
• Genomics
• Nucleosome positioning
• Synthetic biology

Statement dc:rights

• All Rights Reserved.

Language dc:language.iso

en_US

Handle dc:identifier.uri

https://hdl.handle.net/1794/28082

OAI record oai:identifier

https://scholarsbank.uoregon.edu/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Ascholarsbank.uoregon.edu%3A1794%2F28082